demonstration in yeast of the function of bp-80, a putative plant

The Plant Cell, Vol. 13, 781–792, April 2001, www.plantcell.org © 2001 American Society of Plant Physiologists

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

David Humair, Doramys Hernández Felipe, Jean-Marc Neuhaus, and Nadine Paris

1

Laboratoire de Biochimie, rue E. Argand 9, BP2, CH-2007 Neuchâtel, Switzerland

BP-80, later renamed VSR

PS-1

, is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacu-ole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR

PS-1

characteristics andin vitro binding properties described so far favored its receptor function, this function remained to be demonstrated.Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar re-ceptor, Vps10p. By expressing VSR

PS-1

together with GFP fused to the vacuolar sorting determinant of petunia proaleu-rain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR

PS-1

is ineffective on GFP either alone orwhen fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR

PS-1

therefore inter-acts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport ofthe reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR

PS-1

receptorfunction but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent.

INTRODUCTION

The plant secretory system is far less easy to study than itsyeast counterpart, mainly due to the lack of an equivalentmutant collection. Plant and yeast cells, in opposition tomammalian cells, do not use the mannose 6-phosphate–based lysosomal sorting tag (Kornfeld, 1992); instead, thesorting information is carried by a peptidic sequence. Inyeast, the two vacuolar proteins carboxypeptidase Y (CPY;Johnson et al., 1987) and proteinase A (Klionsky and Emr,1989) both are produced with an N-terminal propeptide re-sponsible for the vacuolar location of the mature protein. Al-most 50 mutants defective for vacuolar protein sorting (

vps

)have allowed the identification of proteins involved in theyeast vacuolar sorting pathway. One of these proteins,Vps10p, plays a central role because it is the vacuolar re-ceptor for CPY (Marcusson et al., 1994). Vps10p also is ableto send foreign or malfolded proteins to the vacuole for deg-radation in a process that is believed to be independent ofany sorting signal (Hong et al., 1996).

In plants, two main types of vacuolar sorting determinants(VSDs) have been well studied and are believed to corre-spond to two separate vacuolar sorting pathways. The firsttype could be defined as a sequence-specific VSD (ssVSD)and has been well studied for barley aleurain (Holwerda etal., 1992) and sweet potato sporamin A (Matsuoka andNakamura, 1991). For these two examples, the VSD is lo-

cated within an N-terminal propeptide and contains a con-served tetrapeptide NPIR, the Ile being essential (Matsuokaand Nakamura, 1999). The position of this category of VSDseems to be less important than its sequence specificity, asshown with sporamin A (Koide et al., 1997). The secondtype of VSD is found in C-terminal propeptides (Ct-VSD); itis rather short with no obvious sequence conservation butneeds to be accessible from the C terminus of the propro-tein. The best-studied examples were found in tobaccochitinase A (Neuhaus et al., 1991) and barley lectin(Bednarek and Raikhel, 1991). There is a third type of VSDthat is far less characterized and internal to the polypeptide(reviewed in Neuhaus and Rogers, 1998).

Little is known about the pathway that uses Ct-VSDs be-sides its higher sensitivity to wortmannin, a phosphatidyl-inositol 3-kinase inhibitor, in the BY2 tobacco cell line(Matsuoka et al., 1995). In contrast, a good candidate for avacuolar sorting receptor involved in an ssVSD-associatedpathway has been identified. This putative receptor, BP-80(binding protein of 80 kD), was first isolated from pea clath-rin-coated vesicles (CCVs) by its ability to bind proaleurainVSD in vitro. This binding was pH sensitive because it oc-curred at neutral pH and was abolished at pH 4.0. Bindingassays also showed the specificity of interaction betweenBP-80 and its ligand, because the related ssVSD fromsporamin A was partially able to compete with aleurain VSD,but barley lectin Ct-VSD was not (Kirsch et al., 1994). BP-80is a type I protein with its N-terminal domain involved in rec-ognizing the proaleurain VSD (Kirsch et al., 1994). BP-80was further characterized and cloned (Paris et al., 1997). In

1

To whom correspondence should be addressed. E-mail [email protected]; fax 41-(0)32-7182201.

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782 The Plant Cell

addition to CCVs, it is localized in dilated ends of the Golgiapparatus, a possible plant

trans

-Golgi network, as well asin small structures next to the central vacuole that areassumed to be prevacuoles (Paris et al., 1997). Severalhomologs were identified in different species, including Arabi-dopsis, rice, and maize. BP-80 therefore was renamedVSR

PS-1

(vacuolar sorting receptor from

Pisum sativum

num-ber 1; Paris et al., 1997).

Although all of its characteristics favor its role as a vacuolarsorting receptor, the function of VSR

PS-1

has not been proven,and such a demonstration is always a challenge. Here, we setup an in vivo test in yeast to demonstrate the vacuolar sortingand targeting functions of VSR

PS-1

. In a yeast strain deficientfor the main vacuolar sorting receptor, Vps10p, we expressedVSR

PS-1

together with green fluorescent protein (GFP) fusedto various VSDs. In this mutant yeast, our reporter accumu-lated in the vacuole exclusively when it was fused to theproaleurain VSD and expressed together with the putative re-ceptor VSR

PS-1

. This not only demonstrated that VSR

PS-1

in-teracts with aleurain VSD in vivo but also that this interactionleads to transport of the reporter through the yeast secretorysystem to the vacuole. This assay therefore provides astraightforward tool to address the specificity of interactionbetween VSR

PS-1

and its ligand in vivo, and we already havefound that the plant receptor has a more restricted set ofligands than its yeast equivalent Vps10p.

RESULTS

GFP Is an Appropriate Reporter in Which to Study Plant VSDs in Yeast

It has been reported that a potentially secreted form of GFPwas sent to the vacuole in wild-type yeast (Kunze et al.,

1999). To determine whether this was also the case in astrain with a disrupted gene for the yeast vacuolar receptorVps10p (

D

vps10), we transformed this mutant yeast with thetwo control constructs represented in Figure 1, a secretedGFP (Sec-GFP) and a GFP fused to the yeast CPY vacuolarsignal (CPY–GFP).

The potentially secreted form of the reporter, Sec-GFP,was expressed in a

D

vps10 mutant strain, and the intracellu-lar accumulation pattern of fluorescence was analyzed andcompared with that found in a wild-type yeast cell. The re-sulting transformed strains were observed using a confocalmicroscope, as shown in Figure 2A (Sec-GFP). The wild-type yeast strain showed GFP fluorescence (green) accu-mulating intracellularly, meaning that our reporter is effi-ciently produced and fluoresces in yeast. The subcellularlocation of the reporter (green) can be clearly identified asvacuolar by using a dye specific for the vacuolar membrane,FM 4-64 (red). For more clarity, a set of four images is pre-sented from the same cells, the third one representing amerged and pseudocolored image of GFP in green and FM4-64 in red. Unlike the wild type, the

D

vps10 strain (Figure2A, Sec-GFP) was not able to retain the reporter Sec-GFPintracellularly because no GFP-linked fluorescence was ac-cumulated in these cells. This result clearly showed that theabsence of fluorescence in the mutant strain is the result ofVPS10 disruption. We then assessed by immunoblot (Figure2B) the ratio of GFP found in the medium to GFP retainedwithin the cell for both strains. Some Sec-GFP was clearlyfound in the cell fraction (c) both from a wild-type and a mu-tant strain (

D

vps10), but the amount of GFP was significantlylower when the gene for Vps10p was disrupted. This findingindicated, as described previously (Kunze et al., 1999), thatGFP is sent to the vacuole in wild-type yeast, and our re-sults additionally demonstrated that this is due to Vps10pactivity. This is also in accord with previous observationsshowing a low specificity for Vps10p that can send to thevacuole for degradation many proteins that are either for-eign to yeast or not properly folded (Hong et al., 1996).

We similarly tested how the yeast vacuolar determinantfrom CPY was handled by yeast when fused to GFP. We ex-pressed the construct CPY-GFP (Figure 1) in both the wild-type and

D

vps10 strains and observed the cells by confocalmicroscopy, as shown in Figure 2A (CPY-GFP). As ex-pected, the CPY-GFP was found predominantly in the cen-tral vacuole of the wild-type yeast because the GFP-fluorescent structures (green) were clearly surrounded bythe vacuolar membrane marker (red). In contrast with thewild type, the mutant strain (

D

vps10) no longer accumulateda detectable amount of CPY-GFP in the vacuole. These re-sults were reinforced by immunoblot analysis (Figure 2B). Asseen on the CPY-GFP immunoblot, a significant proportionof reporter protein was secreted (m) by mutant cells(

D

vps10) compared with wild-type cells. The amount ofCPY-GFP still found intracellularly by

D

vps10 cells (c) mayrepresent a form of the reporter on its way out of the cellwith a uniform and weak fluorescent pattern under confocal

Figure 1. Constructs Used in This Study.

Sec-GFP, the secreted form of GFP in which the Arabidopsis chiti-nase B signal peptide (s.p.) was fused to GFP5 (GFP); CPY-GFP, thesignal peptide and the N-terminal propeptide from the yeast CPY(detailed) were fused to GFP5; GFP-Chi, the Ct-VSD of the tobaccochitinase A (detailed) was fused to the secreted form of GFP (Sec-GFP); aleu-GFP, the CPY signal peptide was fused with petuniaaleurain VSD (detailed) to the N terminus of GFP5. For comparison(in parentheses), the corresponding sequence of the previously de-scribed VSD from barley proaleurain (Holwerda et al., 1992) isshown. Underlined amino acids are essential for vacuolar targeting.

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A Plant Receptor Functions in Yeast 783

microscopy, as is seen occasionally in some yeast. Mostimportantly, GFP did not accumulate in any specific subcel-lular location, including the vacuole.

Although confocal microscopy leads to comparable GFPimages for CPY-GFP and Sec-GFP expressed in yeast, im-munoblot analysis (Figure 2B) showed that the reporter wasmore efficiently retained inside the wild-type cells when itcarried the additional CPY vacuolar signal. This could meanthat there is a signal-specific mechanism linked to the pres-ence of the CPY determinant in addition to the retentionmechanism involved in sending foreign or malfolded pro-teins to the vacuole. This finding suggests that, like the CPYvacuolar signal, any potential vacuolar determinant will beexposed properly to the sorting system when fused to theGFP reporter. In addition, the fact that neither Sec-GFP norCPY-GFP was found in the vacuole of the mutant strainshowed that there is no targeting machinery, independent ofVps10p, that will send GFP to the vacuole. Therefore, thesecontrol constructs allowed us to conclude that (1) GFP canbe used as a reporter for vacuolar sorting in a yeast strainthat lacks a functional Vps10p, and (2) GFP should properlypresent a given VSD to its appropriate receptor.

The Petunia Aleurain Propeptide Is Sufficient, in Plants, to Target GFP to the Vacuole

We wanted to test two types of plant signals that are pre-sumably representative of two different vacuolar sortingpathways. The tobacco chitinase A VSD is sufficient to sendGFP5 to a neutral pH vacuole in tobacco protoplasts (DiSansebastiano et al., 1998). We also wanted to use a repre-sentative of the sequence-specific type of signal, the barleyaleurain VSD (Holwerda et al., 1992). In barley proaleurain,the VSD is composed of 24 amino acids organized in threesubgroups, each of which is able to partially redirect a nor-mally secreted protein to the vacuole in protoplasts from to-bacco-derived suspension cells. Unfortunately, becausecodon use preferences are very different between cerealsand yeast, we had to replace the well-described barley aleu-rain VSD with an equivalent coding sequence from the dicot

Figure 2.

Vps10p-Dependent Vacuolar Localization of Sec-GFPand CPY-GFP in Yeast.

(A)

The living cells were visualized with a confocal microscope after

24 hr of induction. The constructs, either a secreted form of GFP(Sec-GFP) or a GFP fused to the yeast vacuolar signal from CPY(CPY-GFP), were expressed in wild-type cells (WT) or in a yeast dis-rupted for the gene encoding the vacuolar receptor Vps10p(

D

vps10). Each condition combines four images from the same cells:from left to right, GFP fluorescence, FM 4-64 staining of a vacuolarmembrane, a pseudocolored merged image with GFP in green andFM 4-64 in red, and the transmitted light image. Bars

5

5

m

m.

(B)

After 24 hr of induction, the transformed cells were separated intwo fractions: the cell extract (c) and the medium (m). Both extractsthen were immunolabeled with anti-GFP antibodies. Molecularmasses of standards are indicated to the left in kilodaltons.

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784 The Plant Cell

petunia. We took amino acids 1 to 44 from petunia pre-proaleurain that should contain the vacuolar sorting infor-mation equivalent to its barley counterpart. Figure 1 showsan alignment of the petunia sequence (aleu-GFP, detailed)and barley proaleurain VSD (in parentheses). Both se-quences are highly homologous and contain the tetrapep-tide NPIR that has been shown to be essential for targeting.Nevertheless, we had to show that this petunia aleurain se-quence was sufficient for vacuolar sorting in plants by usingGFP as a reporter. In our laboratory, we had already usedthe whole propeptide (119 amino acids) from barley aleurainfused to GFP and found that the reporter accumulated in anacidic vacuole (Di Sansebastiano et al., 2001). This necessi-tated the use of a more stable and brighter variant of GFP5,called GFP6, which differs from GFP5 by the mutationsF64L and S65T described previously (Cormack et al., 1996).

For transient expression in plants therefore, we fused GFP6to the first 44 amino acids from petunia preproaleurain, in-cluding the signal peptide and the potential VSD. We trans-formed tobacco leaf protoplasts with the GFP chimera andobserved the fluorescence by using a confocal microscopeafter 24 hr of transient expression, as shown in Figure 3. In

many cells, we found aleu

Ph

-GFP6 fluorescence (Figure 3A,green) accumulated in the large central vacuole that occupiesalmost all of the cell volume (Figure 3B). This vacuolar loca-tion is easy to distinguish in cytoplasmic staining because ininternal confocal sections the latter is limited to a thin layer atthe periphery of the cell (Di Sansebastiano et al., 1998). Inaddition to the large central vacuole, occasional small GFP-labeled structures were visible that may represent interme-diate transport compartments (Figure 3, cell at bottom).This result clearly showed that within petunia preproaleu-rain, the 24 amino acids that follow the signal peptide aresufficient for vacuolar sorting in plants, as are their barleyhomologs.

VSR

PS-1

Redirects the GFP Reporter to the Yeast Vacuole When Fused to Petunia Aleurain VSD

Because we found that GFP5 is a suitable reporter for vacu-olar targeting studies in a

D

vps10 yeast, we fused it witheach of two representative VSDs we chose from plants (Fig-ure 1), tobacco chitinase A (GFP-Chi) and petunia aleurain(aleu-GFP). As shown in Figure 4, both constructs were effi-ciently expressed by a wild-type strain, because the re-porter was visible and accumulated in the vacuole, as incontrols. As for the secreted form of GFP, the fluorescencewas no longer seen intracellularly when the same two fusionproteins with plant signals were expressed in the mutantstrain (

D

vps10). This means that the two plant VSDs did notlead to intracellular retention of our reporter in yeast in theabsence of Vps10p. The aleurain chimera was producedand tested with the signal peptide of yeast CPY (as shownhere) or with the petunia aleurain’s own signal peptide withidentical results (data not shown).

In

D

vps10 yeast cells transformed by each of the fourGFP fusion proteins described previously, we coexpressedthe sequence coding for the putative plant vacuolar sortingreceptor, VSR

PS-1

. As shown in Figure 5A, this coexpressiondid not lead to any retention of Sec-GFP, CPY-GFP, orGFP-Chi. In contrast, VSR

PS-1

clearly affected aleu-GFP dis-tribution because the reporter accumulated in the lumen ofthe vacuole (Figure 5A, aleu-GFP

1

VSR

PS-1

). The vacuolarlocation of the GFP (green) was clearly confirmed using FM4-64 to stain the vacuolar membrane (red). The exclusivevacuolar location of aleu-GFP was even more visible in theGFP channel alone (far left). All of the confocal images pre-sented were taken under the same conditions by using iden-tical laser power for GFP excitation. Therefore, the GFP-linked fluorescences shown here were directly comparableto each other, meaning that a similar amount of aleu-GFPaccumulated in the vacuole due to coexpression with eitherVSR

PS-1

or Vps10p function (Figures 5A and 4, respectively).The percentage of

D

vps10 cells that were fluorescent whenaleu-GFP and VSR

PS-1

were coexpressed reached 60%,which is close to the 70% of fluorescent wild-type cells trans-formed with the same aleu-GFP. Within this GFP-labeled

Figure 3. Demonstration of the Vacuolar Sorting Properties of thePetunia Aleurain Propeptide in Tobacco Leaf Protoplasts.

Living protoplasts expressing aleuPh-GFP6 were visualized using aconfocal microscope 24 hr after transformation. Two cells areshown after illumination with either the dual fluorescein isothiocyan-ate/tetramethylrhodamine isothiocyanate fluorescence filter (A) ortransmitted light (B). The fluorescence image uses the attributedpseudocolors green for GFP and red for chloroplast autofluores-cence. Bar in (A) 5 10 mm.

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population of

D

vps10 cells, 67% accumulated aleu-GFP in avacuolar pattern. The other, much less abundant pattern isdiffuse, similar to aleu-GFP alone in a

D

vps10 strain andmay be due to a lack of coexpression of VSR

PS-1

in thosecells. This high percentage of aleu-GFP redirected to theyeast vacuole because of the putative plant receptor sug-gests that VSR

PS-1

is as efficient as Vps10p in terms of vac-uolar sorting and targeting.

As shown in Figure 5C, we performed an immunoblotanalysis after 48 hr of induction. At this stage, the level ofexpression decreased due to the exhaustion of galactose;therefore, much of the reporter should have reached its finaldestination. Figure 5B shows the GFP pattern observed un-der confocal microscopy after this longer culture time. Thefluorescence accumulation pattern is identical to that foundat 24 hr, with even less residual nonvacuolar labeling. Onthe immunoblot at this stage, we detected less of the highermolecular weight transport intermediates than after shorterculture times (data not shown). The predominant form thatwe observed at 48 hr (Figure 5C, aleu-GFP) is similar in sizeto native GFP, suggesting that the VSD has been removed.This immunoblot analysis confirmed a significant intracellu-lar retention of the reporter when aleu-GFP and VSR

PS-1

were coexpressed in a

D

vps10 strain. Compared with thesignal obtained for a wild-type cell expressing the samealeu-GFP construct (left lane), VSR

PS-1

–mediated retention isan efficient process. In the negative control represented byGFP-Chi

1

VSR

PS-1

, the GFP signal found intracellularly wasalmost undetectable, and a longer exposure time was re-quired to make it visible (Figure 5C, bottom). Most impor-tantly, the presence of this residual GFP-Chi was not due tothe presence of VSR

PS-1

because a similar amount of re-porter was retained as well in a mutant yeast cell.

Because the NPIR motif was shown to be a major compo-nent of the barley aleurain VSD (Holwerda et al., 1992) and tobe essential for the sporamin VSD (Matsuoka and Nakamura,1999), we performed a preliminary experiment to investigatethe importance of the NPIR motif from the aleurain VSD inour assay. The Ile therefore was replaced with a glycine inthe aleu-GFP chimera. When coexpressed with the plant re-ceptor, we found an obvious diminution in the percentage ofcells with a GFP-labeled vacuole as well as an overall dimi-nution in intensity compared with the original aleu-GFP(data not shown). Although we could not express our resultsin a quantitative way, this finding nevertheless suggestedthat the sequence specificity feature of the aleurain VSD isas essential in our yeast assay as it has been shown to be inplant cells. The fact that we did obtain an intermediate an-swer between negative and positive control cells also is inagreement with previous work showing that the barley aleu-rain VSD with all four NPIR amino acids mutated was stillable to target 25% of aleurain to the vacuole in tobacco cul-ture cells (Holwerda et al., 1992).

The effect of VSR

PS-1

therefore is linked exclusively to thepresence of the ssVSD, as in petunia proaleurain, becausethe plant receptor does not retain the reporter when it carries

Figure 4. Vps10p-Dependent Vacuolar Localization of GFP-Chi andaleu-GFP in Yeast.

The living cells were visualized with a confocal microscope after 24hr of induction. The constructs, either a GFP fused to the tobaccochitinase A VSD (GFP-Chi) or the petunia aleurain VSD fused to GFP(aleu-GFP), were expressed in wild-type cells (WT) or a yeast dis-rupted for the gene encoding the vacuolar receptor Vps10p(Dvps10). Each condition combines four images from the same cellsas described for Figure 2A. Bars 5 5 mm.

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786 The Plant Cell

a different type of plant VSD (GFP-Chi) and is less efficientwhen the essential NPIR sequence is mutated. The fact thatVSR

PS-1

did not affect Sec-GFP repartition also suggeststhat VSR

PS-1

is different from Vps10p in its ability to targetforeign or misfolded proteins to the yeast vacuole.

VSR

PS-1

Apparently Traffics within the Yeast Secretory System Like Its Equivalent Vps10p

To assess where the plant receptor accumulated in theyeast cell, we performed immunolabeling, as shown in Fig-ure 6. First, we labeled cells expressing the plant receptorwith antibodies directed against VSRs (Figure 6A). The dot-like labeling pattern for VSR

PS-1

is absent from an untrans-formed cell (Figure 6C, inset), and when merged with atransmitted light image it appears next to the vacuole (Fig-ure 6A, both). This labeling shows that our plant receptordoes not accumulate in the vacuolar membrane or the vacu-ole even when overexpressed. For comparison, we per-formed similar immunolabeling on wild-type yeast cells tolocalize the native Vps10p. As shown in Figure 6B, we alsoobtained dot-like labeling similar to what has been de-scribed previously (Cooper and Stevens, 1996), and again itwas very close to the vacuolar membrane. This resultsuggests that in a mutant yeast strain the plant vacuolar re-ceptor is located in compartments similar to those accumu-lating Vps10p in wild-type cells. We then performed doubleimmunolabeling on wild-type strains expressing the plant

Figure 5.

The Expression of VSR

PS-1

Specifically Restores the Vacu-olar Location of aleu-GFP in the Absence of the Yeast VacuolarSorting Receptor Vps10p.

(A)

The living cells were visualized with a confocal microscope after24 hr of induction. The

D

vps10 strain was doubly transformed withthe plant putative vacuolar sorting receptor VSR

PS-1

and one of thefour GFP fusion constructs. These GFP chimera carry no vacuolarsignal (Sec-GFP), the CPY yeast vacuolar signal (CPY-GFP), the to-bacco chitinase A VSD (GFP-Chi), or the petunia aleurain VSD (aleu-GFP). Each condition combines four images from the same cells asdescribed for Figure 2A. Bars

5

5

m

m.

(B)

Effect of VSR

PS-1

on aleu-GFP localization in

D

vps10 cells after48 hr of induction. Shown are (top) a fluorescence image of a

D

vps10 cell expressing both VSR

PS-1

and aleu-GFP (green) andstained for the vacuolar membrane with FM 4-64 (red) and (bottom)the same cell in transmitted light. Bar

5

5

m

m.

(C)

Immunoblot analysis after 48 hr of induction by using anti-GFPantibodies on a cell extract from either wild-type yeast (WT) or ayeast disrupted for the gene encoding the vacuolar receptor Vps10p(

D

vps10) and expressing the GFP reporter fused to either the petu-nia aleurain (aleu-GFP) or the chitinase A (GFP-Chi) VSD in the pres-ence (

1

) or absence (

2

) of VSR

PS-1

. The top and bottom panelsrepresent the same blots exposed for either 5 or 15 sec, respec-tively. Molecular masses of standards are indicated to the left in kilo-daltons.

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receptor (Figure 6C). When merged (both), the two imagesclearly showed that all of the VSR

PS-1

–labeled compart-ments also labeled for the yeast receptor (arrowheads). Thisshows that the plant receptor is able to accumulate in thesubcellular location where its yeast equivalent is concen-trated in the latter case, as a result of controlled cycling be-tween the late Golgi and the prevacuole.

These results suggest that the accumulation of aleu-GFPin the yeast vacuole was not simply the result of one-waycotransport of the plant receptor together with its ligandfrom the Golgi to the vacuole. They also suggest that at

least part of the overexpressed plant receptor may be ableto use the yeast sequence-specific trafficking machinery.

DISCUSSION

VSR

PS-1

was the first putative vacuolar sorting receptor iso-lated and cloned from a plant based on its hypotheticalfunction. It binds in vitro to the VSD of barley aleurain andto related ssVSDs but not to a Ct-VSD from barley lectin(Kirsch et al., 1994). Two criteria for the receptor functionare now fulfilled by our results: (1) the petunia aleurain VSD,sufficient for vacuolar targeting in plants, is recognized invivo by VSR

PS-1

; and (2) this interaction causes targeting ofthe reporter aleu-GFP to the vacuole in

D

vps10 yeast. The invivo demonstration of VSR

PS-1

function added to its previ-ously described properties, (1) its pH-dependent binding(Kirsch et al., 1994) similar to the mammalian mannose6-phosphate system (Kornfeld, 1992) and (2) its subcellularlocation in plant cells (Paris et al., 1997), allow us to proposethe following model (Figure 7). At the level of the

trans

-Golgi(circled 1), where the pH is believed to be neutral or slightlyacidic compared with that in the mammalian system, VSR

PS-1

would sort proaleurain (gray squares) from other solubleproteins (white squares). More precisely (Figure 7, detail),the sorting would occur by interaction of the N-terminal do-main of VSR

PS-1

with the ssVSD carried by proaleurain(aleu.). Simultaneously, VSR

PS-1

would expose its C-terminaldomain to cytosolic proteins such as adaptins that are in-volved in CCV formation. The complex of VSR

PS-1

and pro-aleurain then would be transported (circled 2) to an acidiccompartment, the prevacuole. Due to the decrease of pH,the ligand then would be released from VSR

PS-1

(circled 3).Finally, we propose that the free receptor would be recycledback to the Golgi (circled 4) although it is not fully demon-strated by our data. The shuttle vesicles used by the recep-tor must be clathrin coated at least in one direction becauseVSR

PS-1

originally was purified from this type of vesicle(Kirsch et al., 1994).

The fact that VSR

PS-1

functions in yeast is an argument infavor of conserved vacuolar targeting machineries betweenyeast and plants. VSR

PS-1

must meet its ligand and thereforemust accumulate to some extent in the compartment wherevacuolar sorting occurs in yeast, presumably the late Golgi(Graham and Emr, 1991). Interestingly, immunostaining ofVSR

PS-1

always colocalized with Vps10p structures, andmost importantly, the plant receptor did not show any vacu-olar accumulation. These results support the hypothesisthat VSR

PS-1

, like Vps10p, traffics from the late Golgi to theprevacuolar compartment where aleu-GFP is released andtherefore does not migrate from the late Golgi together withits ligand all the way to the vacuole. However, we cannotfully rule out that the plant receptor is sent to the yeast vac-uole where it is rapidly degraded, and one still may arguethat the high level of expression of VSR

PS-1

may lead to a

Figure 6. The Plant Vacuolar Sorting Receptor VSRPS-1 Is Not Vacu-olar and Colocalizes with Vps10p in Yeast Cells.

Cells were fixed after 24 hr of culture and immunolabeled with anti-VSR and/or anti-Vps10p antibodies. The resulting immunostainingwas visualized using a confocal microscope.(A) A Dvps10 cell expressing VSRPS-1 was immunolabeled with anti-VSR antibodies (left). A transmitted image of the same cell (trans.)was taken and merged to the immunostaining image (both).(B) A wild-type cell was immunolabeled with anti-Vps10p antibodies(left). A transmitted image of the same cell (trans.) was taken andmerged to the immunostaining image (both).(C) A wild-type cell expressing VSRPS-1 was immunolabeled withanti-VSR and anti-Vps10p antibodies. The immunolabeling imageswere pseudocolored in green for VSRPS-1 and red for Vps10p andmerged (both). The inset shows the background staining with anti-VSR antibodies on untransformed wild-type cells. Arrowheads markthe location of double-labeled compartments.Bars 5 1 mm.

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788 The Plant Cell

constant and sufficient amount of the plant receptor withinthe late Golgi with no recycling requirement.

It has been shown that, when overexpressed, the yeastvacuolar receptor Vps10p devoid of its cytosolic domainstill sent 35% of CPY to the vacuole in a

D

vps10 strain

(Cooper and Stevens, 1996). Yet, these authors alsoshowed that, at a level of expression similar to the wild-type receptor, no residual activity was found for the sametruncated receptor. In our overexpressing context, we havepreliminary results suggesting that the cytosolic domain ofthe plant receptor is not necessary for traffic in yeast (datanot shown), as was described in plants (Jiang and Rogers,1998). The fact that Vps10p and VSR

PS-1

colocalize in vivoin yeast can be interpreted as evidence for some recyclingin yeast in addition to a constant flow of transport, althoughwe would need a lower expressing system to demonstratethis hypothesis. This led to the exciting suggestion thatplant trafficking signals in the cytosolic tail of VSR

PS-1

couldrecruit some of the yeast proteins involved in vesicle forma-tion and, more precisely, in retrograde transport to the lateGolgi. The C-terminal domains of VSR

PS-1

and Vps10p areextremely different in length as well as in sequence, exceptfor a tyrosine motif that is known to interact with adaptinsfrom CCVs. It will be interesting to pursue this comparisonto identify crucial trafficking motifs that may be shared byyeast and plant cells. It also would be interesting to dis-cover the pathway actually used by VSR

PS-1

in yeast to de-termine, for example, which type of adaptin complex isrecruited. Two secretory pathways have been shown tosend proteins to the yeast vacuole: one for soluble proteinssuch as CPY, mediated by Vps10p, Pep12p, and AP1adaptins, and the other for membrane proteins such as al-kaline phosphatase (Stack et al., 1995). The latter pathwayis independent from Vps10p and Pep12p and uses AP3adaptins (Odorizzi et al., 1998).

Although our results suggest strong similarities betweenyeast and plant vacuolar trafficking machineries, we founddifferences in ligand preferences between the respectivereceptors. Unlike Vps10p, VSR

PS-1

recognizes neither theGFP itself as a foreign protein nor the yeast vacuolar signalfrom CPY. This indicates a divergence between the twosorting systems in terms of their affinity for VSDs. We canexpect such a difference because plants have developed amuch more complex vacuolar system, having in some cellstwo (Paris et al., 1996) if not three (Jauh et al., 1999) differ-ent destinations for soluble vacuolar proteins. The Vps10pdomain involved in ligand recognition may contain twobinding sites, one for sequence-specific interactions andthe other for unfolded structures (Jørgensen et al., 1999).Structural requirements for VSRPS-1 ligand binding havebeen published recently (Cao et al., 2000). The authorsalso found indications for two possible recognition sites inVSRPS-1, one specific for the NPIR motif and another definedas a “non-NPIR site.” Our assay would provide a usefultool to address in vivo the structural features of the N-ter-minal domain of VSRPS-1.

The ligand specificity for VSRPS-1 found using in vitro ap-proaches (Kirsch et al., 1994) is now corroborated in vivo byour yeast assay. VSRPS-1 is clearly specific for the type ofVSD carried by aleurain and does not recognize tobaccochitinase A VSD, a typical C-terminally required signal. The

Figure 7. Model for VSRPS-1 Function in Plants.

The trans-Golgi (left) is the region of the secretory pathway wheresorting of vacuolar soluble proteins is believed to occur. At this neu-tral pH, the plant vacuolar sorting receptor (brackets) sorts (step 1)proaleurain (gray squares) from other soluble proteins (whitesquares). The detail shows more precisely the structure of VSRPS-1,with its N-terminal domain (N) interacting with the ssVSD carried byproaleurain (aleu.). It also shows the C-terminal domain (C) of VSRPS-1

on the cytosolic side of the membrane that is available for contactswith proteins such as adaptins that are involved in CCV formation.The complex of VSRPS-1 and proaleurain (step 2) is packed intotransport vesicles and targeted to an acidic compartment (right). Thedecrease in pH causes the ligand to be released from VSRPS-1 (step3) within the acidic compartment, which is possibly a prevacuole.The free receptor then recycles back to the Golgi (step 4), again bymeans of transport vesicles. We know that CCVs are used by the re-ceptor in plants to shuttle between the Golgi and an acidic compart-ment, but we do not know in which direction of transport. Thismodel is based on our demonstration of VSRPS-1 function in yeastand on previously published characteristics (Kirsch et al., 1994;Paris et al., 1997).

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reporter GFP can efficiently expose N-terminal, C-terminal,and even internal peptides (Abedi et al., 1998) and remainsfluorescent. Therefore, we can study the interaction be-tween potential signals and VSRPS-1 in a more realistic con-text than with synthetic peptides linked to an affinitycolumn. The range of ligands that are recognized by VSRPS-1

is an important issue because confusing results implicate ei-ther this receptor or members of its growing family of ho-mologs. For example, the VSR homologs PV72 and PV82from pumpkin recognize two peptides within pro 2S albu-min, one internal and one C-terminal (Shimada et al., 1997).The ssVSD of proaleurain is clearly able to release the dou-blet at 72 and 82 kD that was retained on a column expos-ing the internal sequence of 2S albumin, which means thatthese VSR homologs from pumpkin are able to recognizethe two types of sequences. Unfortunately, the internal se-quence of pro 2S albumin used in that study has not beendemonstrated to be used as a vacuolar signal in the contextof the native protein. VSRPS-1, or a homolog, also recognizesthe vacuolar targeting signal carried at the C terminus of 2Salbumin in vitro (Kirsch et al., 1996). Finally, some tobaccoVSRs interact with the C-terminal region of a Nicotiana alataprotease inhibitor that is necessary for vacuolar targeting(Miller et al., 1999).

In addition to these VSRs for which ligand affinity was in-vestigated directly, there are other VSR homologs for whichwe have no direct evidence in terms of vacuolar sorting func-tion besides the high sequence similarities (Ahmed et al.,1997; Paris et al., 1997). The Arabidopsis VSR family is nowrepresented by at least seven members, and it is not knownwhether all variants have ligand specificities and subcellularlocation identical to VSRPS-1 (Paris and Rogers, 1996). To-gether with the original receptor, two variants of VSRPS-1 alsowere cloned from the same cDNA library made from pea seed(Paris et al., 1997). It is troubling, therefore, that only one pro-tein was isolated based on its ability to bind in vitro to theproaleurain VSD. So far, there is no available tool, such as an-tibodies, that would allow one to identify specifically each ofthese VSR variants in planta, which may explain why contra-dictory localization results have been published. For example,the same Arabidopsis VSR homolog was found either mainlyin a PEP12 prevacuolar compartment (Sanderfoot et al., 1998)or almost exclusively in the plasma membrane (Laval et al.,1999). To date, the studies on VSR variants subcellular loca-tion are puzzling as well, and at this stage it is premature andtherefore risky to conclude that all of the VSRs function ex-actly like VSRPS-1. It seems more reasonable to postulate thateach VSR has a different spectrum of ligands and perhapseven functions in different pathways.

Our assay is now providing a clear and fast in vivo meansto investigate each VSR variant in terms of ligand specificitywithin the limits imposed by the differences between theyeast and plant vacuolar systems. We will need a method toquantitate vacuolar targeting efficiency if we want to com-pare the possible differences in affinity between differentmembers of the VSR family.

METHODS

Strains and Media

Bacterial strains were grown on standard media (Miller, 1972). Yeaststrains were grown on yeast extract, peptone, dextrose medium, orsynthetic dextrose (SD) medium supplemented as necessary(Sherman et al., 1979). The yeast strains SEY6210 (MATa leu2-3,112 ura3-52 his3-D200 trp1-D901 lys2-801 suc2-D9) and EMY3(SEY6210 vps10D1::HIS3) have been described previously (Robinsonet al., 1988; Marcusson et al., 1994).

Constructs

All of the constructs used were made according to basic methodsdescribed previously (Sambrook et al., 1989). The yeast–Escherichiacoli shuttle vector pGAL was used throughout this study (Blum et al.,1989). Two derivatives of pGAL were produced: (1) pGAL-BglII wasobtained by removing the BglII fragment containing the TRP1 gene,and (2) pYWG1 was obtained by removing the NsiI-PstI fragmentcontaining a part of the URA3 gene. For plant transient expression,we used the vector pGY1, which contains 35S promoter and termi-nator sequences (Neuhaus et al., 1991). Green fluorescent protein(GFP) constructs were derived from the previously described plas-mids pSGFP5 and pSGFP5T (Di Sansebastiano et al., 1998).

For yeast expression, the construct containing the secreted GFP(Sec-GFP; Figure 1) was obtained by introducing the BamHI-SacIfragment from pSGFP5, which encodes the Arabidopsis thalianachitinase signal peptide fused in-frame with the GFP5 sequence, inthe corresponding sites of pYWG1. The construct containing theGFP fused to the C-terminal propeptide of tobacco chitinase A (GFP-Chi; Figure 1) was made by cloning the BamHI-SacI fragment frompSGFP5T, which encodes Sec-GFP5 with the C-terminal addition ofthe tobacco chitinase vacuolar sorting determinant (VSD), intopYWG1. For the GFP5 fused to the signal peptide and the propeptidefrom carboxypeptidase Y (CPY-GFP; Figure 1), we amplified by poly-merase chain reaction (PCR) a fragment encoding the first 50 aminoacids of preproCPY by using a plasmid encoding the CPY-invertasefusion protein (pLJL2; a kind gift from E. Marcusson, University ofCalifornia–San Diego, La Jolla) as a template and two primers gener-ating a BamHI site in 59 and a NheI site in 39. The PCR fragment thenwas used to replace the BamHI-NheI fragment from Sec-GFP5, giv-ing CPY-GFP5. To obtain the fusion between GFP5 and the propep-tide of Petunia hybrida aleurain (aleu-GFP; Figure 1), we first createda maxiprimer containing the first 20 amino acids of CPY (signal pep-tide) by using pLJL2 as a template and two primers generating aBamHI site in 59 and a sequence homologous with codons 21 to 25of the petunia preproaleurain in 39. This maxiprimer then was used in59 to amplify codons 21 to 44 from preproaleurain by using pETH3(Tournaire et al., 1996; GenBank accession number U31094) as atemplate and a second primer adding an NheI site in 39. This PCRfragment then was used to replace the BamHI-NheI fragment fromSec-GFP, giving the construct aleu-GFP.

The construct containing VSRPS-1 was obtained by introducing the2-kb XmaI-KpnI fragment from NP471 corresponding to the full cod-ing sequence (Paris et al., 1997; GenBank accession numberU79958) in the equivalent sites of pGAL-BglII. All constructs werecontrolled by sequencing.

For expression in tobacco protoplasts, the first 44 amino acids of

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790 The Plant Cell

preproaleurain from petunia were placed in front of a more stableand brighter derivative of GFP5, called GFP6, which contains themutations F64L and S65T (Cormack et al., 1996). We first generateda BamHI site in 59 and an NheI site in 39 of the first 44 codons of pe-tunia preproaleurain by PCR using pETH3 as a template. We thencloned the resulting fragment with the NheI-SacI GFP5 fragmentfrom CPY-GFP between the BamHI and SacI sites of pGY1. We laterintroduced the two point mutations within the GFP5 sequence byPCR to give the more stable GFP6. The resulting construct, calledaleuPh-GFP6, is cloned between the 35S promoter and terminator se-quences carried by pGY1.

Cell Labeling and Confocal Microscopy

Transformed or cotransformed yeast cells were grown for 24 hr at308C in selective SD medium containing 2% galactose for induction.We then stained specifically the yeast vacuolar membrane by usingthe lipophilic styryl dye FM 4-64 (N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide; Molecu-lar Probes, Eugene, OR; Vida and Emr, 1995). The cells were incu-bated for 15 min at 308C in inducing SD medium containing 8 mM FM4-64 and then rinsed for 90 min at 308C in inducing SD medium.These incubation times are sufficient for the dye to reach the vacu-olar membrane with no residual plasma membrane or endosomalstaining. Cells were immobilized on a slide in 0.4% low-melting-pointagarose for observation with a microscope.

Tobacco (Nicotiana tabacum) protoplasts were isolated fromleaves of cv SR1 (Nagy and Maliga, 1976). A polyethylene glycol–based transformation protocol then was used (Negrutiu et al., 1987;Freydl et al., 1995; Di Sansebastiano et al., 1998), and the trans-formed protoplasts were visualized 24 hr after transformation.

Images were collected with a Leica (Wetzlar, Germany) confocallaser microscope by using a Leica TCS 4D operating system. Either afluorescein isothiocyanate or a tetramethylrhodamine isothiocyanatefilter set was used to detect GFP or FM 4-64, respectively. Digital im-ages were pseudocolored using Adobe Photoshop version 4.0.1(Mountain View, CA), with red attributed to FM 4-64 and green attrib-uted to GFP. An image with transmitted light also was collected us-ing the confocal microscope.

Immunostaining was performed on Dvps10 or wild-type cells grownovernight in inducting conditions when transformed with VSRPS-1.Cells were fixed using a modified version of the standard protocol(Pringle et al., 1991). The cells then were fixed in their growing me-dium for 1 hr with 3.7% formaldehyde at 308C and for at least 12 hr atroom temperature with 0.1 M KPO4, pH 6.4, containing 3.7% para-formaldehyde. They were washed three times in buffer alone andonce with buffer A (1.2 M sorbitol, 0.12 M K2HPO4, and 33 mM citricacid, pH 5.9). The cell wall was digested using b-glucuronidase at10,000 units/mL (slug juice; Sigma) and 5 mg/mL zymolyase 20T(ICN Biomedical, Costa Mesa, CA) in buffer A for 75 min at 308C. Af-ter washing, the membranes were treated with 0.5% Triton X-100 for5 min, and immunolabeling was performed as described previously(Paris et al., 1996). The primary antibodies used were the monoclonal14G7 anti-VSR antibodies at a 1:100 dilution (Paris et al., 1997) andthe anti-Vps10p serum (a gift from Scott Emr, University of Califor-nia–San Diego, La Jolla) at a 1:100 dilution. The secondary antibod-ies used were anti-mouse rhodamine Red-X conjugated or anti-rabbit fluorescein conjugated (catalog numbers 115-295-100 and711-095-152, respectively; Jackson ImmunoResearch, West Grove,PA) at a dilution of 1:100.

Protein Gel Blot Analysis

Transformed yeast cells were incubated at 308C in inducing SD me-dium containing 2% galactose to an OD600 of 5 to 10. Cells equiva-lent to 5 OD600 units were collected and separated from the culturemedium by centrifugation. The cell pellet was resuspended in SDSgel sample buffer (Laemmli, 1970), frozen in liquid nitrogen, andboiled for 10 min at 1008C. Freezing and boiling were repeated twice.The crude cellular extract then was centrifuged at 14,000g for 10 minto remove debris. Proteins were precipitated from the medium with10% trichloroacetic acid for 15 min at 2208C and then recovered bycentrifugation (10,000g, 30 min). The trichloroacetic acid pellet waswashed twice in acetone and resuspended in 20 mL of SDS gel sam-ple buffer. The cell extract and the proteins from the medium (bothequivalent to 5 OD600 units) were loaded on a 12% SDS-PAGE gel.Immunoblots were prepared as described previously (Towbin et al.,1979) using anti-GFP antibodies (diluted 1:5000; Molecular Probes)and secondary goat anti-rabbit antibodies (diluted 1:30,000) coupledto alkaline phosphatase (Sigma). The bands then were visualized us-ing the IMMUN-STAR chemiluminescent protein detection system(Bio-Rad).

ACKNOWLEDGMENTS

We thank Prof. S.D. Emr for advice and for the gift of the yeast strainsand Antje Heeze-Peck for advice on the visualization of vacuoles inyeast. We thank S. Marc-Martin and Dr. G.-P. Di Sansebastiano fortheir valuable help. This work was supported by a European Molecu-lar Biology Organization long-term fellowship to N.P. (57-1996) andby a Swiss National Research Foundation grant (3100-46926.96).

Received October 23, 2000; accepted January 26, 2001.

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